Photochemical behavior of Safranine-Riboflavin complex in the degradation of folic acid

Article abstract of DOI:10.1016/j.jphotochem.2014.07.010

The photochemistry of safranine complexed with riboflavin and the photoinduced electron transfer from folic acid to the complex were investigated by steady-state and time resolved absorption spectroscopy, and the photoproducts were further analyzed by LC-orbitrap-FT-MS. UV-vis spectra of the complex showed a decrease in the safranine band at 520 nm with increasing concentration of riboflavin, which was attributed to complex formation. A 1:1 (safranine:riboflavin) stoichiometry was determined using Job's method. Following safranine photoexcitation, there is formation of a radical pair in the complex by donation of an electron from ground-state riboflavin to excited-state safranine. The cation radical of riboflavin within this complex reacts with folic acid to yield FA^+?, which may accept an electron from the anion radical of safranine, avoiding the photobleaching of the safranine. In contrast, the reaction of the anion radical of non-complexed safranine with folic acid leads to safranine photobleaching.

Full text of DOI:10.1016/j.jphotochem.2014.07.010

Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 32–39
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Photochemical behavior of Safranine-Riboflavin complex in the
degradation of folic acid
Willy G. Santos∗, Regina S. Scurachio, Daniel R. Cardoso
Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São Carlense, 400 CP 780, 13560-970, São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 April 2014
Received in revised form 2 July 2014
Accepted 19 July 2014
Available online 1 August 2014
The photochemistry of safranine complexed with riboflavin and the photoinduced electron transfer from
folic acid to the complex were investigated by steady-state and time resolved absorption spectroscopy,
and the photoproducts were further analyzed by LC-orbitrap–FT–MS. UV–vis spectra of the complex
showed a decrease in the safranine band at 520 nm with increasing concentration of riboflavin, which
was attributed to complex formation. A 1:1 (safranine:riboflavin) stoichiometry was determined using
Job’s method. Following safranine photoexcitation, there is formation of a radical pair in the complex
by donation of an electron from ground-state riboflavin to excited-state safranine. The cation radical of
riboflavin within this complex reacts with folic acid to yield FA+•, which may accept an electron from the
anion radical of safranine, avoiding the photobleaching of the safranine. In contrast, the reaction of the
anion radical of non-complexed safranine with folic acid leads to safranine photobleaching.
© 2014 Elsevier B.V. All rights reserved.
Keywords:
Dyes
Riboflavin
Safranine
Folic acid
Photochemistry
Electron transfer
1. Introduction
In recent developments of pharmaceuticals, some organic func-
tional dyes have been shown considerable potential for drug
Safranine is a dye, classified as phenazine derivative, which is
widely used in the textile, pharmaceutical, paper and cosmetic
industries [1,2]. In addition, safranine finds application in many
areas of research, such as biological stain [3,4], electropolymer film
synthesis [5,6], and clinical and medicinal application as bio-probe
for determination of hyaluronic acid, inhibition of human ribonu-
cleotide reductase, detection of double stranded DNA, molecular
adhesive for protein immobilization, as probe for membrane orga-
nization, and in several other application [7,8]. This molecule is
also employed as a photosensitizer for various chemical reactions
[9–11].
In the presence of appropriate molecule, safranine in its triplet-
state may accept or donate electron to form the reduced or oxidized
species, respectively. The electron transfer between dyes and other
redox active compounds is especially suited to studies with model
membranes and micelles [12–14]. These reactions play an impor-
tant role in understanding the molecular mechanism of drug
toxicity and the effect of the presence of dyes in cell membrane
[15–20].
development [21]. In particular, photosensitizer fluorochromes
with low toxicity and high photochemical activity, such as flavins,
have been widely used in tumor treatment [22] and inactivation of
virus in plasma [23]. Riboflavin (Rib) is a relevant photosensitive
fluorochrome which is also known as water-soluble Vitamin B2. In
biochemistry, flavin molecules may interact easily with biological
macromolecule due to its planar structure. For instance, riboflavin
is used as fluorescent probe in the analysis of deoxyribonucleic acid
(DNA) [12,24,25].
Electron-transfer reactions from excited-states of dyes repre-
sent an important route to produce radicals that might induce
various chemical or biological processes. Therefore, the choice of
appropriate components for participating in such electron trans-
fer reactions is important, and is a subject of continuous research.
After efficiently receiving or donating an electron, these com-
pounds should be able to form reactive radicals or radical ions,
either directly or through fragmentation. However, the interaction
of safranine and riboflavin was not investigated in detail. The pho-
tochemical study with these two sensitizers is important because
they can be developed as therapeutic agents.
Concerning the dyes repair mechanism, this work report the
electron transfer mechanism of isolated dyes and the com-
plexed dyes with folic acid. The photosensitized mechanism of
safranine–riboflavin complex toward folic acid and the dyes repair
were studied in detail by direct observation of the transient species
∗
Corresponding author. Tel.: +55 1633739976.
E-mail addresses: willy glen@yahoo.com.br, willygsantos@gmail.com
(W.G. Santos).
http://dx.doi.org/10.1016/j.jphotochem.2014.07.010
1010-6030/© 2014 Elsevier B.V. All rights reserved.

W.G. Santos et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 32–39
33
OH
O
O
OH
O
N
O
NH
N
N
CH3
CH3
HN
O
Cl
H2N
H3C
NH2
CH3
N
N
NH
N
OH
OH
HO
N
O
OH
HN
N
NH2
Safranine chloride (Sf)
Riboflavin (Rib)
Folic acid (FA)
Scheme 1. Chemical structure of the compounds studied.
[Fe(phenanthroline)3]2+ formed after addition of phenanthroline.
This procedure was accomplished by adding 1 mL of photolyzed
sample, 0.5 mL of acetate buffer (600 mL of 1 mol L−1 sodium
acetate and 360 mL 0.5 mol L−1 H2SO4 diluted to a total vol-
ume of 1 L), and 2 mL of a 0.1% aqueous phenanthroline solution
to 10 mL of water. After standing for 15 min in the dark, the
amount of [Fe(phenanthroline)3]2+ was determined by measuring
the absorbance at 510 nm (ε = 11,100 L mol−1 cm−1). The procedure
was repeated with a sample of ferrioxalate kept in the dark. Five
independent runs were averaged to determine the intensity of light
incident on the sample. The absorbance measurements were car-
ried out employing a Hitachi U-3501 (Hitachi-Hitech, Tokyo, Japan)
spectrophotometer.
and their reaction kinetics using time resolved laser flash photo-
lysis technique. The aim of this study was to gain further insight
into the photosensitized mechanism of the complex with promis-
ing applications for photoinduced electron transfer in biological
system.
2. Experimental
2.1. Chemicals
Safranine
O
97% (3.7-diamino-2.8-dimethyl-5-phenylphe-
nazinium chloride), riboflavin (Vitamin B2) and folic acid 97%
(Pteroyl-l-glutamic acid or Vitamin B9) were purchased from
Sigma-Aldrich (Steinheim, Germany) and used without further
purification. Stock solutions of safranine (10−2 mol L−1
)
and
2.4. Liquid chromatography accurate ultra-high resolution mass
spectrometry
riboflavin (10−3 mol L−1) were prepared in buffer solution at 298 K
and pH 7.4 (Scheme 1).
LC–MS/MS analyses were carried out using an LTQ Orbitrap
Velos FT-MS mass spectrometer (Thermo Fischer Scientific, Bre-
men, Germany) with a UPLC Thermo Scientific model Accela LC
Systems. 5 ␮L of the sample was injected onto a Waters Spherisorb
S5 C18 reverse phase column (2 mm × 250 mm × 4.6 ␮m) main-
tained at 40 C. A constant flow rate of 0.3 mL min−1 was used with
the following gradient: 15% B isocratic for 20 min, then up to 90%
B for 10 min and 15% B for 5 min. Solvent A was 0.1% formic acid in
water and solvent B was 0.1% formic acid in acetonitrile. The final
composition was analyzed by direct infusion into an HESI interface
ionization (Thermo Scientific, Bremen, Germany).
2.2. Spectroscopic measurements
Absorption spectra at different temperatures were recorded
on a Multskan Go Reader (Thermo Scientific, Waltham MA,
US) spectrometer using a 1 × 1 cm quartz cuvette. In photolysis
experiments, the absorption was taken at different irradiation
times at 298 K, using a light emitting diode as a light source at
532 nm 25 nm filtered by an interferometer at 520 nm 5 nm.
Steady-state fluorescence measurements were made on a
Hitachi model F4500 (Hitachi-Hitech, Tokyo, Japan) spectrometer
equipped with a thermostat for the quenching studies at different
temperatures.
3. Results and discussion
Transient absorption spectra were obtained using a LFP-112
Luzchem nanosecond laser flash photolysis spectrometer (Luzchem
research, Ottawa, Canada). Excitation of the samples was per-
formed with the third harmonic (355 nm) of a Brio Nd-YAG laser
(Quantel, Les Ulis, France), with 5.2 ns light pulses and power of
10 mJ cm−2. Kinetic analyses were made with the Luzchem propri-
etary software.
3.1. Ground-state and singlet-state process
The complex between riboflavin (Rib) and safranine (Saf) was
first studied by steady-state UV–vis absorption spectroscopy.
Fig. 1a shows the absorption spectrum of riboflavin that presents
absorption maxima at 373 nm and 440 nm. The safranine absorp-
tion has a maximum centered at 520 nm, which is decreased upon
addition of riboflavin.
2.3. Potassium ferrioxalate actinometry
The Job’s plot shown in Fig. 1b presents the ratio of
absorbance between the riboflavin measured at 373 nm and
safranine at 520 nm at different volume ratios, considering the
The absorbance of 0.15 mol L−1 solution of K3[Fe(C2O4)3] was
measured at 550 nm, measured before and after the solution was
irradiated for 20 min at 20 ◦C, to determine the fraction of light
absorbed. The number of moles of ferrous ion produced upon
light irradiation was then determined by measuring the amount of
concentration of each stock solutions ([Sf] = 1 × 10−5 mol L−1
;
[Rib] = 1 × 10−4 mol L−1) at different proportional volumes. The
absorbance ratio initially increases upon complex formation and

34
W.G. Santos et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 32–39
2000
2.5
2.0
1.5
1.0
0.5
0.0
(a)
[Safranine] = 1X10 mol L + [Riboflavin]
Sf (1X10-5 mol L-1) + Rib (1X10-5 mol L-1
(a)
)
Temperature
328 K absence of Rib
328 K
1300
1200
1100
1000
1500
1000
500
0
318 K
308 K
298 K
Isosbestic Point
520 nm
300
310
320
330
Temperature (K)
200
300
400
500
600
700
500
550
600
650
700
750
λ (nm)
λ (nm)
0.010
0.008
0.006
0.004
0.002
0.000
(b)
1.30
1.25
1.20
1.15
1.10
1.05
1.00
30.5
30.0
29.5
29.0
28.5
28.0
Sf 1:1 Rib Complex
(b)
Temperature
278 K
283 K
288 K
298 K
308 K
0.39
0.40
0.41
0.42
0.43
0.44
1000 / RT
10/0 9/1
8/2
7/3
6/4
5/5
4/6
3/7
2/8
1/9 0/10
VRib / VSaf (volume/ volume)
0
2
4
6
8
10
Fig. 1. (a) Absorption spectra of safranine in the presence and absence of riboflavin.
(b) Job’s plot for the Sf–Rib complex in aqueous buffer solution at pH 7.4 and 298 K.
[Riboflavin] X 10-6 ( mol L-1 )
Fig. 2. (a) Fluorescence spectra and (b) Stern–Volmer plot for Sf + Rib system in
aqueous buffer solution at pH 7.4 and at different temperatures. Inset (a): Fluo-
rescence intensity of safranine at different temperatures. Inset (b): Rate constant
for the quenching of safranine fluorescence recorded at different temperatures.
ꢁexc = 520 nm; ꢁem = 570 nm.
subsequently decreases with further safranine addition. The max-
imum value of absorbance ratio in the Job’s plot (Fig. 1b) shows a
stoichiometry of 1:1 (Rib:Sf) for the dominant species.
The fluorescence emission of safranine has a maximum centered
at 570 nm, which is quenched in presence of increasing concentra-
tion of riboflavin, as may be observed in Fig. 2a and b.
where Ao and Ai are the safranine absorption at 520 nm in the
absence and presence of riboflavin, m and n represent the stoi-
chiometric indices for safranine and riboflavin, respectively. The
extinction coefficient (εo) for safranine in the ground-state is
4.8 0.23 × 104 L mol−1 cm−1 and d is the optical path of cell used
for absorbance measurements.
The high fluorescence quenching constant (1kq = 2.86
0.21 × 1012 L mol−1 s−1) at 298 K is faster than the diffusion con-
trolled rate, and suggest that the process is likely to be dominated
by a static quenching rather than a dynamic quenching. In fact, in
agreement with this measurements using time resolved fluores-
cence measurements were carried and no changes were observed
for the safranine time-decay (ꢀF = 1.40 0.02 ns at 298 K). However,
in the situation of complex formation, fast fluorescence quenching
may be possible and proportional to a complex quantity. Fig. 2b
inset shows the fluorescence quenching constant of safranine in
presence of riboflavin recorded at different temperatures.
Since the complex (Sf–Rib) does not present any absorption in
the region of the safranine spectrum, the concentration of complex
may be calculated in terms of the depletion of safranine absorption
at 520 nm, see Fig. 1a. Assuming [Sf–Rib] < [Sf] or [Rib], we may
write the following equation as
To evaluate the coefficients m and n coefficients in Eq. (1), data
from the measurement of the formation constant of the complex
were used. The values of both m and n are nearly unit, consider-
ing a 1:1 stoichiometry for the complex. The equilibrium constant
value (Keq = 1.6 0.1 × 104 L mol−1 at 298 K) was determined in
high concentrations of riboflavin, assuming that the concentra-
tion of riboflavin does not change significantly upon complexation,
since Rib ꢀ Sf . The effect of temperature on the equilibrium is
[
]
[ ]
shown in Fig. 3.
From the angular coefficient in Fig. 3 (inset) and the Van’t Hoff
equation (Eq. (2)), the enthalpy of complex formation can be calcu-
lated, and has the value ꢂH◦ = 50.5 0.8 kJ mol−1. The same value
of ꢂH◦ was found for all three curves in Fig. 3 (inset).
m
n
Ao − A = Keq × εo × d × Sf × Rib
(1)
[
]
[
]
i

W.G. Santos et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 32–39
35
0.10
0.08
0.06
0.04
0.02
0.00
0.04
safranine (1X10-5 mol L-1) + riboflavin
safranine 1X10-5 mol L-1
(a)
810 nm
5 X10-5 mol L-1
0.03
1 X10-5 mol L-1
650 nm
400 nm
2 X10-6 mol L-1
0 mol L-1
300 nm
0.02
0.01
0.00
-0.01
-0.02
-0.03
-0.04
-0.05
Time delay
0.2 μs
7.7 μs
20.3 μs
78.0 μs
300
305
310
315
320
300
400
500
600
700
800
900
Temperature (K)
λ (nm)
Fig. 3. Difference spectra for safranine absorption in the absence and presence of
riboflavin at different temperatures. Inset: Van’t Hoff plot for the equilibrium of
safranine–riboflavin complexation in aqueous buffer at pH = 7.4. ꢁobs = 520 nm.
0.03
0.02
Sf (1X10-5 mol L-1) + Rib (2X10-5 mol L-1)
(b)
650 nm
ꢂSo
R
ꢂHo
RT
400 nm
360 nm
ꢀ
ꢁ
810 nm
slow
ln Keq
=
−
(2)
slow
fast
slow
0.01
The value of ꢂS◦ was estimated from the intercept point, relat-
ing values <4 J K−1 mol−1 for all curves, which is strongly dependent
on the free safranine and riboflavin in solution. In agreement
with the thermodynamic values obtained for absorption mea-
surements, the enthalpy found for fluorescence measurements
at 570 nm, in Fig. 2 (inset), has ꢂH◦ = 48.7 0.7 kJ mol−1 and
0.00
λ
= 810 nm
-0.01
-0.02
-0.03
-0.04
Time delay
5.1 μs
33.3 μs
98.0 μs
320.0 μs
ꢂS◦ = 9 0.4 J K−1 mol−1
The value of Gibbs free energy was calculated from the following
equation:
.
ꢀ
ꢁ
ꢃGo = −RT ln Keq
(3)
300
400
500
600
700
800
900
The high negative value of Gibbs free energy (−50.1 kJ
1.2 mol−1) compared with entropy indicates a favorable formation
of Sf–Rib complex for all concentration of safranine and riboflavin
and that the formation is mainly due to hydrophobic interaction.
λ (nm)
Fig. 4. Transient absorption spectrum of safranine in aqueous solution at pH 7.4 and
recorded at different times after laser pulse, in the (a) absence and (b) presence of
riboflavin. Inset: Observed pseudo first-order rate constant as function of riboflavin
concentration. ꢁexc = 532 nm and power of 16 mJ cm−2
.
3.2. Triplet state process
In the presence of riboflavin, the triplet state of safranine is
quenched, and, as observed 98 ␮s after the laser pulse, there
is, simultaneously, an increase in the absorption at 360 nm and
absorption depletion at 400–420 nm and 600–700 nm, indicating
an increase in the amount of semireduced form of safranine and
within the same lifetime range as has been observed with safranine
in the presence of other electron donors [11,27]. This may sug-
gests that electron transfer between riboflavin and safranine triplet
state, yield the semireduced form of safranine and the semioxidized
cation radical of riboflavin.
Following excitation with a 532 nm laser pulse in deaereated
buffer solution at pH 7.4, safranine shows transient absorptions
centered at 300, 400, 650 and 810 nm, as shown in Fig. 4a. No trans-
ients are observed in air saturated solutions. At pH 7.4, the major
peak of the triplet state of safranine may be seen at 810 nm, and is
observed over the whole spectral range to decay with the lifetime
of 18 ␮s at 298 K.
In the absence of quenchers the triplet excited state decays by
a first order kinetics due to a self-quenching process [10,11,26,27],
leading to long lifetime absorption at 400 nm and 650 nm that are
attributed to the triplet and semireduced form of the safranine with
a lifetime of 100–200 ␮s at 298 K in aqueous buffer pH 7.5. The
semioxidized form of safranine is observed at 440 nm with lifetime
of 30–40 ␮s, and is formed by the same self-quenching reaction
3Sf + Rib → Sf−• + Rib+•
The radical cations of various flavins has been ascribed previ-
ously by P.F. Heelis and co-workers to have absorption bands in the
300–450 nm and 600–650 nm regions, and to decay by first order
kinetics, with k1 = 103 s−1 and 104 s−1 at pH = 4 and 9, respectively
[28]. This rate constant is in agreement with our species observed
at 360 nm with k1 = 2.47 0.03 × 103 s−1 at 298 K and pH 7.5. Fig. 5
shows the kinetic decays of the various transient species.
3Sf + Sf → Sf+• + Sf−•
The lifetime and absorption band of the semireduced and oxi-
dized radical of safranine were observed in our previous work
[10,11].
In this self-quenching mechanism, it is very difficult to distin-
guish the semioxidized safranine kinetics from the triplet–triplet
decay due to very similar lifetimes.
The second-order rate constant for the quenching of 3Saf by Rib
(3.3 0.1 × 108 L mol−1 s−1) was determined from safranine life-
time at 810 nm (Fig. 4b inset). This is lower than the diffusion limit

36
W.G. Santos et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 32–39
0.020
Sf (1
X
10-5 mol L-1) + Rib (2
X
10-5 mol L-1)
Sf (1 x10-5 mol L-1) + FA
0.014
0.012
0.010
0.008
0.006
0.004
0.002
0.000
0.008
360 nm
0.006
0.004
0.002
0.000
0.015
0.010
0.005
0.000
(400 nm)
(360 nm)
0
20
40
60
80
100
Time (μs)
(810 nm)
absence of Rib
0
100
200
300
400
0
20
40
60
80
100
Time (μs)
Time (μs)
Fig. 5. Kinetic traces for different transient decays monitored in real-time at various
wavelength following laser pulses of 10 mJ cm−2 at 532 nm in argon-saturated buffer
solution at pH 7.4 and 298 K. Inset: Triplet-decay of safranine and transient growth
of the riboflavion cation radical, both observed at 360 nm.
Fig. 6. Kinetic traces for triplet-excited safranine decay monitored in real-time at
810 nm following laser pulses of 10 mJ cm−2 at 532 nm for increasing concentrations
of folic acid in argon-saturated buffer solution at pH 7.4 and 298 K. Inset: observed
pseudo first-order rate constant as function of folic acid concentration.
in aqueous solution (1010 L mol−1 s−1), in contrast with what is fre-
quently observed with bimolecular deactivation of triplet-states.
In the presence of folic acid, the triplet of safranine is quenched
with lifetime of 100 ␮s, and is accompanied by an increase in
absorption in the 400–450 nm region and other at 600–700 nm.
These absorptions have been ascribed to semireduced form of safra-
nine [11,27], which is formed by an electron transfer from the folic
acid to the safranine triplet state.
Only Saf (1
X
10-5 mol L-1)
0.020
0.015
0.010
0.005
0.000
-0.005
FA + Saf (1
X
10-5 mol L-1) + Rib (2 10-5mol L-1)
X
absence of FA
5X
8X
1X
10-5 mol L-1
10-5 mol L-1
10-4 mol L-1
3Sf + FA → Sf−• + FA+•
The addition of folic acid to safranine solution quenches the
triplet absorption of non-complexed safranine with kq = 4.29
0.08 × 109 L mol−1 s−1 at 298 K (Fig. 6). The lifetime of the cation
radical of folic acid is very short, and it rapidly decomposes to
form secondary products (pterin-6-carboxylic, p-aminobenzoyl-l-
glutamic acid and the oxaziridine derivative) [29–32].
0
100
200
300
400
Time (μs)
When the folic acid is added in the Saf–Rib complex solution, the
rate of growth of the cation radical absorption at 360 nm is slower
than with Saf–Rib complex alone, as may be seen in Fig. 7.
The differences in the rate of growth of the transient suggest
complex competitive process of quenching of safranine by FA and
by riboflavin, which is characterized by an increase in the solution.
The low depletion of the absorption supports this idea, since reac-
tion of the cation radical of riboflavin with FA should deplete all the
absorption of the radical species, which is not observed in Fig. 7 at
high concentrations of FA.
Fig. 7. Kinetic traces for radical cation formation of riboflavin monitored in real-time
at 360 nm following laser pulses of 10 mJ cm−2 at 532 nm for increasing concentra-
tions of folic acid in argon-saturated buffer solution at pH 7.4 and 298 K.
3.3. Photobleaching process
The photolysis of the Sf–Rib system was carried out by excit-
ing the safranine absorption band at 520 nm, in degassed aqueous
buffer pH 7.4 solutions. Fig. 8a and b shows the absorption behavior
Table 1
Photobleaching rate constant for systems containing safranine, riboflavin and folic acid in aqueous buffer solution at 298 K and pH 7.4.
[Safranine] (mol L−1
)
[Riboflavin] (mol L−1
)
[Folic acid] (mol L−1
)
kPB × 102 min−1
˚
PB
1 × 10−5
3 × 10−5
5 × 10−5
1 × 10−5
1 × 10−5
1 × 10−5
1 × 10−5
1 × 10−5
1 × 10−5
1 × 10−5
1 × 10−5
–
–
–
–
–
–
–
–
–
1.01
1.12
1.41
1.22
1.65
2.38
2.85
1.90
0.82
0.03
<10−3
0.026
0.029
0.036
0.031
0.042
0.061
0.078
0.049
0.021
0.001
–
5 × 10−6
1 × 10−5
3 × 10−5
–
1 × 10−6
1 × 10−6
5 × 10−6
1 × 10−5
5 × 10−5
3 × 10−5
3 × 10−5
3 × 10−5
3 × 10−5

W.G. Santos et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 32–39
37
10A − 1
1.0
0.8
0.7
0.5
0.3
0.2
0.0
Sf (1X X
10-5 mol L-1) + Rib (1 10-5 mol L-1)
(a)
ln
= −ε × Io × d × ˚PB × t = kPB × t
(4)
10A − 1
o
Irradiation Time (min)
0
where
˚
PB
is the photobleaching quantum yield,
A
is the
30
absorbance due to the complex absorption, Ao is the safranine
absorption in the absence of riboflavin, ε is the molar absorption
coefficient at the irradiation wavelength, d is the irradiation path
length and Io is the light source intensity at 520 nm.
From the slopes of the plots in Fig. 8c, and using Parker ferriox-
alate actinometer for the determination of the incident light flux
(Io = 8.68 × 108 E min−1), the photobleaching quantum yields were
determined and compared for different chemical systems, as shown
in Table 1. It may be seen that the presence of riboflavin or FA in the
safranine solution increases the photobleaching of the safranine.
However, when folic acid is added to the Sf–Rib complex solution,
the safranine photobleaching is decreased and its proportional to
the folic acid concentration.
60
90
120
145
175
205
239
269
300
400
500
600
700
λ
(nm)
The more effective photobleaching observed for Sf/FA in com-
parison with the Sf–Rib complex supports the results obtained for
quenching of the triplet-state of safranine, where the FA is seen to
quench safranine ten times faster than Sf–Rib complex.
The proposed mechanism, considering all photochemical stud-
ied in this work, is presented in Scheme 2.
1.0
10-5 mol L-1) + Rib (3
X
10-5mol L-1) + AF (1 10-5 mol L-1)
X
(b)
Sf (1
X
0.9
0.7
0.6
0.4
0.3
0.1
0.0
Irradiation Time (min)
0
30
60
105
145
179
209
269
329
389
3.4. Photoproducts of folic acid sensitized by safranine
Photoreaction was studied of mixtures containing safranine
(5 × 10−5 mol L−1) and folic acid (5 × 10−4 mol L−1) in aqueous
phosphate buffer solution (pH 7.4, I = 0.1 mol L−1 NaCl at 298 K).
High resolution accurate mass spectra, recorded in the positive ion
mode, show characteristic profiles consisting of peaks attributable
to safranine, folic acid and three main photoproducts derived
from folic acid. These photoproducts display different reten-
tion times (1) tr = 5.49 min ([M + H]+ = 192.05112 m/z 2.5 ppm,
as calculated for C7H6O2N5 and assigned as 6-formylpterin);
300
400
500
600
700
λ
(nm)
(c)
(2) tr = 5.87 min ([M + H]+ = 267.09717 m/z
1.4 ppm, as cal-
0.0
culated for C12H15O5N2 and assigned as p-aminobenzoyl-
glutamate moiety released from the pterin), and (3) tr = 7.68 min
([M + H]+ = 456.12506 m/z 2.5 ppm, as calculated for C19H18O7N7
and assigned as a folic acid oxaziridine derivative). The same
photoproducts obtained for Sf/FA chemical system have previ-
ously been observed by Scurachio and co-workers [29], in the
riboflavin is used as sensitization agent for folic acid degradation
[34–44].
Results with Sf–Rib complex in presence of folic acid show
the same peaks as in Sf/FA system. However, in the equilibrium
situation, the riboflavin and safranine in non-complex make it
impossible to identify the effective participation of the complex
in folic acid degradation.
-0.5
-1.0
-1.5
-1
1X10-5 mol L
)
Only Sf
(
-1
-1
3X10-5 mol L
)
FA + Sf
(
1X10-5 mol L
-1
)
+ Rib
(
0 mol L
-1
-1
-1
-1
-6
1X10 mol L
-6
5X10 mol L
-5
1X10 mol L
-5
5X10 mol L
The photoproducts of the photolysis of Saf–Rib/FA system
at 532 nm for 1 h30 min were characterized and identified
by high-resolution accurate mass spectroscopy in the posi-
tive mode. The photoproducts were tentatively assigned to
6-formylpterin, p-aminobenzoyl-l-glutamic acid and oxaziridine
derivative, respectively. These same mass spectrometry charac-
terizations were obtained by others researchers upon irradiating
riboflavin in the presence of folic acid [29–32]. The proposed mech-
anism of the photoproducts generation by complex sensitization is
shown in Scheme 3.
0
10
20
30
40
50
60
Time (min)
Fig. 8. Absorption spectra of (a) safranine/riboflavin and (b) safra-
nine/riboflavin/folic acid systems after different times of irradiation; (c)
photobleaching of safranine alone and in complex form, in the presence and
absence of folic acid at 298 K and pH = 7.4.
of the safranine/riboflavin and complex/FA solutions in differ-
ent times of irradiation. The kinetic properties were studied by
monitoring the absorption band of the safranine at different irra-
diation times. The rate of photobleaching is given by Eq. (4)
[11,33]
The products of the safranine and complex-induced photooxi-
dation of folic acid corroborate to understand the nature of photo
reaction and identify a new photochemical method to avoid the
riboflavin degradation.

38
W.G. Santos et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 32–39
(Complex)
Keq
KISC
[1(Sf) Rib]*
...
[3(Sf) Rib]*
hν
...
(Sf)o + Rib
[(Sf)o Rib]
...
(810 nm)
knr
h
ν
k -et
1ko
ket
1(Sf)
KISC
3(Sf)
.
+
.
[(Sf)- Rib
]
...
3kq[O2]
Photobleaching
(400 nm)
+ FA
+ FA
+
.
.
[(Sf)- Rib] + FA
+
.
.
...
(Sf)- + FA
(400 nm)
(420 nm)
ksq[Sf]
Major Photoproducts :
* Pterin-6-carboxylic
-
.
+
.
(Sf) + (Sf)
(420 nm)
(450 nm)
* p-aminobenzoyl-L-glutamic
* Folic acid Arylnitrone derivative
Photobleaching
Scheme 2. Proposed photochemical mechanism for Sf–Rib complex reactions in the presence and absence of FA.
Complex
[(Sf) Rib]
...
h
ν
Folic acid
Imine
oxaziridine derivative
Folic Acid (FA)
[(Sf)3 Rib]
...
NH2
O
NH2
NH2
O
O
O
NH2
ket
+
.
-.
[(Sf)
Rib
]
...
+
.
NH
N
NH
N
N
N
N
-e / -H+
Ox.
O
N
N
N
N
O
O
O
O
N
N
NH
N
N
NH
NH
HN
N
NH2
-.
[(Sf)
Rib ]
...
NH2
NH2
NH2
H2O
Isomerization
NH2
O
OH
H
O
N
OH
O
OH
O
N
N
O
O
Ox.
N+
+
O
N
NH
O-
O
N
N
H
HN
N
NH2
N
NH2
O
N
Pterin-6-carboxylic
NH2
HN
N
p-aminobenzoyl-L-glutamic
NH2
Folic acid
arylnitrone derivative
Major Photoproducts
Scheme 3. Proposed mechanism for the generation of folic acid photoproducts.

W.G. Santos et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 32–39
39
4. Conclusion
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Absorption and fluorescence spectra indicate the forma-
tion of
a complex between safranine and riboflavin in the
ground-state with stoichiometry 1:1 (Rib:Sf) for the dominant
species. The equilibrium constant and others thermodynamic
parameters (Keq = 1.6 0.1 × 104 L mol−1, ꢂH◦ = 48.7 0.7 kJ mol−1
,
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accepts an electron from riboflavin, forming the very reactive cation
radical of riboflavin and the semireduced form of the safranine.
The riboflavin cation radical decays by first order kinetics, with
k1 = 2.47 0.03 × 103 s−1. Excitation at 532 nm (in the safranine
absorption band) of deareated solutions of Saf/Rib complex leads to
formation of the safranine triplet state, absorbing at 810 nm, which
decays to form two transient band at 360 nm and 650 nm, attributed
to riboflavin cation radical and the semireduced form of safra-
nine radical, respectively. Folic acid quenches safranine triplet state
by an electron transfer process (kq = 4.29 0.08 × 109 L mol−1 s−1).
When the folic acid is added in the Saf–Rib complex solution,
the lifetime of radical growth is slower than with the Saf–Rib
complex alone, indicating a competitive process between safra-
nine and Saf/Rib to react with FA. This conclusion is supported by
photobleaching experiments, where the increases of the folic acid
concentration decrease the safranine bleaching.
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